Detection of diamines in biological fluids

ABSTRACT

A fast-acting and portable diagnostic kit for detecting diamines in biological fluids is in the form of a diamine oxidase and a chromogenic system to detect the presence of hydrogen peroxide resulting from oxidation of such diamines. The kit may especially be used to detect cadaverine or putrescine or a mixture thereof, for example in a vaginal secretion, in order to detect Gardnerella-related bacterial vaginosis.

This invention relates to a method for the identification of diamines,particularly putrescine and cadaverine in vaginal secretions, and theuse of this in diagnosis of a certain clinical condition, and to adiagnostic kit to enable the method to be applied routinely.

Vaginitis is a widespread problem in general practice. One common formof vaginitis was characterised by Gardner and Dukes (H. L. Gardner andC. D. Dukes, Am. J. Obstet. Gynecol., 1955, 69, 962-976) who described acondition associated with a grey homogeneous discharge having a pH of5.0-5.5 and accompanied by minimal inflammation. The condition appearsto be associated with the organism Gardnerella vaginalis in the presenceof other organisms, notably anaerobic bacteria. This form of vaginitisis now often called bacterial vaginosis to acknowledge the complexity ofits microbiological origin.

Clinical diagnosis of this condition is aided by identification of "cluecells" (vaginal epithelial cells with adherent surface bacteria), araised pH, and a "fishy" odour generated on adding alkali to thesecretion (the "amine" test) (H. L. Gardner and C. D. Dukes, loc. cit.;T. Pheifer et al., New Eng. J. Med., 1978, 298, 1429-1434; R. Amsel etal., Am. J. Med., 1983, 74, 14-22; Report of The Working Group on "TheDiagnosis of Bacterial Vaginosis", Scand. J. Urol. Nephrol. (Suppl.),1984, 86, 260-261).

Microbiological confirmation of G. vaginalis is not straightforward andculture results take 2-3 days. There is no simple, rapid, diagnosticprocedure. It is known that the diamines putrescine and cadaverine occurat significantly elevated levels in Gardnerella-related vaginitis (K. C.S. Chen et al., J. Clin. Invest., 1979, 63, 828-835); these diamines areprobably produced by the symbiotic growth of G. vaginalis in thepresence of the anaerobes. Earlier work showed that the presence of thediamines correlated with clinical symptoms in 96% of patients (K. C. S.Chen et al., J. Infect. Dis. 1982, 145, 337-345).

Previously the detection of the diamines has depended on electrophoresisor thin-layer chromatography (K. C. S. Chen et al., loc. cit.) requiringspecialist equipment and personnel. The aim of this invention is toprovide a method to establish the presence of the diamines in a way thatcan be carried out in a laboratory, clinic, surgery, or in the home, bypersons not skilled in chemical analysis or microbiological procedures.

The invention uses a selected diamine oxidase which reacts with thediamines, putrescine and cadaverine, to give hydrogen peroxide. This isdetected by a colour reaction produced by peroxidase and a suitablechromogenic system. A suitable diamine oxidase (E. C. 1.4.3.6) can beisolated from pea seedlings (R. E. McGowan and R. M. Muir, PlantPhysiol., 1971, 47, 644-649); this oxidises putrescine and cadaverinewith a high specificity. Other diamine oxidases, for example that frompig kidney(Sigma Chemical Co.), can be used in a similar way. Peroxidaseis a well-known enzyme that is commercially available. For thechromogenic system, a number of different reagents may be used. Amixture of 4-aminoantipyrine and 3,5-dichloro-2-hydroxybenzenesulphonicacid, introduced by Barham and Trinder (D. Barham and P. Trinder,Analyst, 1972, 97, 142-145) to detect hydrogen peroxide, proves verysatisfactory, but other phenolic components, for example chromotropicacid or 2,4,6-tribromo-3-hydroxybenzoic acid, may also be usedsatisfactorily. Other chromogenic systems which also work successfullyinclude 3,3',5,5'-tetramethylbenzidine and2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonate).

The diagnostic test in its first form involves taking a vaginal swab andagitating it in a solution containing the diamine oxidase, peroxidase,and the chromogenic compounds. When a mixture of 4-aminoantipyrine and3,5-dichloro-2-hydroxybenzenesulphonic acid is used as the chromogenicsystem, the positive magenta colour develops in under two minutes and isstable for more than one hour. Quantitative measurement can be obtainedfrom the optical density of the solution, but this is not necessary forthe clinical diagnostic test. Negative samples give no colour, or atmost a very pale colour. The solution of reagents for the test in thisform can be made by simple addition of water to a freeze-dried mixtureof all the ingredients prepared in a suitable vessel. This freeze-driedmixture can be stored without deterioration of its efficiency.Alternatively the reagents can be constituted in other ways, for examplein a tablet form or adsorbed on to paper to other surface layer.

Clinical samples of 183 vaginal secretions have been tested and theresults correlated with existing diagnostic procedures-the "amine" test,"clue cells" and culture of G. vaginalis. These correlations, and thesensitivities and specificities, are presented in Example 7 (see below).The diamine test results correlate closely with the microbiologicalculture and with the two sideroom techniques; "clue cell" and "amine"tests. The agreements in the comparisons with "clue cells" and the"amine" test are marginally better than those with the microbiologicalresults from the Public Health Laboratory Service. In all threecomparisons the diamine test gave slightly poorer apparent specificitythan sensitivity. Thus, sensitivity for microbiological, "clue cell" and"amine" tests was 86%, 96% and 93% respectively. The new test thus workswell as a diagnostic procedure.

EXAMPLES EXAMPLE 1 Linearity of Response of the Diamine Oxidase/DyeSystem to Increasing Concentrations of Putrescine

Procedure

Each incubation contained the following reagents:

4-aminoantipyrine (5 mg/ml; 0.1 ml);

3,5-dichloro-2-hydroxy-benzenesulphonic acid (35 mg/ml; 0.1 ml);

peroxidase (1 mg/ml; 0.1 ml);

diamine oxidase (5-15 units/ml; 0.02 ml);

putrescine (0-1.25 mM; 0.1 ml);

buffer (0.1M tris-HCl, pH 7.5; 2 ml).

The reagents were mixed and the absorbance at 515 nm was recorded aftera 10 minute incubation. The absorbance was proportional to the amount ofputrescine added, so establishing that the relationship betweenabsorbance and the quantity of putrescine in the assay was linear overthe range studied (0-125 nmol putrescine per assay).

                  TABLE                                                           ______________________________________                                        nmol putrescine                                                                         A 515 nm    nmol putrescine                                                                           A 515 nm                                    ______________________________________                                        0         0           25          0.292                                       1         0.012       50          0.584                                       2.5       0.028       75          0.885                                       5         0.058       100         1.160                                       7.5       0.088       125         1.446                                       10        0.113                                                               ______________________________________                                    

EXAMPLE 2 Additivity of the Response of the Diamine Oxidase/Dye Systemto the Two Diamines, Putrescine and Cadaverine

The procedure was as described in Example 1, but using either putrescineor cadaverine, or mixtures of both putrescine and cadaverine. Putrescineand cadaverine gave similar absorbances on a molar basis and whenpresent together in the incubation the absorbance produced was thatpredicted by a simple addition of the absorbances given by the twodiamines separately.

                                      TABLE                                       __________________________________________________________________________                Absorbance 515 nm                                                 Substrate concentration                                                                   column 1                                                                              column 2 column 3         column 4                        of each diamine                                                                           putrescine alone                                                                      cadaverine alone                                                                       mixture of putrescine + cadaverine                                                             column 1 + column               __________________________________________________________________________                                                  2                                1 nmol     0.011   0.009    0.018            0.020                            10 nmol    0.113   0.096    0.225            0.209                           100 nmol    1.105   1.104    2.163            2.209                           __________________________________________________________________________     (Column 4 is calculated as the addition of the absorbances in columns 1       and 2.)                                                                  

EXAMPLE 3 Response of the Diamine Oxidase/Dye System to Other Amines orDiamines

The procedure was as described in Example 1, but the putrescine wasreplaced by other biologically important amines. The other aminesincluded: alanine, asparagine, aspartic acid, arginine, cysteine,cystine, glutamic acid, glutamine, glycine, histidine, isoleucine,leucine, lysine, methionine, ornithine, phenylalanine, proline, serine,threonine, tyrosine, tryptophan, valine, 4-aminobutyric acid, histamine,phenethylamine, spermine and tyramine. Colour production was notdetectable, or was slight or very slow to develop with these otheramines. Thus, the response of the diamine oxidase/dye system to thesebiologically important amines that may be present in secretions orfluids could be readily distinguished from the colour production byputrescine and cadaverine.

EXAMPLE 4 Alternative Dye Systems Used in Conjunction with the DiamineOxidase

The procedure was as described in Example 1, except that the incubationcontained 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonate) (14 mg/ml;0.1 ml) instead of 4-aminoantipyrine and3,5-dichloro-2-hydroxybenzenesulphonic acid. The absorbance was measuredat 646 nm after an incubation at room temperature for 60 minutes.

                  TABLE                                                           ______________________________________                                        nmol putrescine                                                                         A 646 nm    nmol putrescine                                                                           A 646 nm                                    ______________________________________                                        0         0           25          0.172                                       1         0.008       50          0.336                                       2.5       0.018       75          0.490                                       5         0.035       100         0.643                                       7.5       0.054       125         0.801                                       10        0.070                                                               ______________________________________                                    

This alternative dye system gave absorbances proportional to theputrescine concentration and thus demonstrated that the procedure wasnot dependent on the dye system quoted in Example 1 but would operatewith other hydrogen peroxide detection systems.

EXAMPLE 5 Stability of the Reagents in a Dried Form

Transparent polystyrene tubes were prepared containing in freeze-driedform the following components:

4-aminoantipyrine (0.5 mg);

3,5-dichloro-2-hydroxybenzenesulphonic acid (3.5 mg);

peroxidase (0.1 mg);

diamine oxidase (0.2 units);

tris-HCl, pH 7.5 (0.2 mmol).

This freeze-dried mixture contained the same amounts of the reagents asin Example 1, with the omission of putrescine.

Each tube was reconstituted with 2.3 ml water after 6 weeks storage at4° C. and the reactivity compared with a fresh solution of the samecomposition. Putrescine (50 nmol) was added to each test (in triplicate)and the absorbances compared.

                  TABLE                                                           ______________________________________                                        Absorbances 515 nm                                                                                    mean                                                  ______________________________________                                        Fresh        0.588  0.587      0.612                                                                              0.596                                     Reconstituted                                                                              0.608  0.607      0.609                                                                              0.608                                     ______________________________________                                    

Thus, when the components were stored in a dried form there was no lossof reactivity by the diamine oxidase/dye system. Similarly, when thesame components were dried on to a strip of Whatman No. 1 chromatographypaper and the paper dipped into a solution containing putrescine, amagenta colour developed.

EXAMPLE 6 Application of the Diamine Oxidase/Dye System to the Analysisof Diamines in Vaginal Secretions

Vaginal swabs were taken from:

(i) patients consulting for vaginitis,

(ii) patients consulting for cervical smears or family planning advice,

(iii) women invited to health checks.

Each swab was washed twice with 1 ml 0.1M-tris/HCl buffer, pH7.5. Anyparticulate material in the combined washings was removed by briefcentrifugation and/or filtration. An aliquot (0.5 ml) was added to amixture containing:

tris/HCl buffer(pH7.5, 0.1M, 1.5 ml);

3,5-dichloro-2-hydroxybenzenesulphonic acid (35 mg/ml, 0.1 ml);

4-aminoantipyrine (5 mg/ml; 0.1 ml);

peroxidase (1 mg/ml; 0.1 ml);

diamine oxidase (10 units/ml, 0.02 ml).

The absorbance at 515 nm was measured after a 10 minute incubation atroom temperature and the amount of diamine calculated from a standardcalibration. From a survey of 183 swabs the amount of diamine in eachswab was estimated. The range of values was found to be 0-540 nmoldiamines/swab.

EXAMPLE 7

183 vaginal swabs were assayed quantitatively by the procedure describedin Example 6 and the results compared with the microbiological cultureby the Public Health Laboratory Service and with current sideroommethods of diagnosing Gardnerella-related vaginitis. Details of the"amine" test, identification of "clue" cells and of the taking, storing,transporting and cultures of swabs at the Public Health LaboratoryService have been described (T.C.O'Dowd et al., J. Roy. Coll. Gen.Pract., 1987, 37, 59-61).

Microbiological Comparison

The Public Health Laboratory Service identified 57 swabs as Gardnerellavaginalis positive and the remaining 126 swabs as Gardnerella vaginalisnegative. The mean quantity of diamine measured per swab for theGardnerella positive group was 161 nmol diamines (+146 nmol s.d.) andfor the Gardnerella negative group was 37 nmol diamines (+86 nmol s.d.).The difference was statistically highly significant.

If a cut-off at 25 nmol diamines/swab is chosen as the dividing linebetween a positive and a negative result in the diamine oxidase/dyesystem assay, the sensitivity of the diamine test was 49/57=86%, i.e. 8apparent "false negatives", and the specificity of the diamine test was100/126=79%, i.e. 26 apparent "false positives".

"Clue Cell" Comparison

171 swabs were evaluated for "clue cells". 52 swabs were described as"clue cell positive", 17 swabs as "clue cell uncertain" and 102 swabs as"clue cell negative". The results of the diamine test were 200 nmoldiamines (+141 nmol s.d.)/swab, 54 nmol diamines (+103 nmol s.d.)/swaband 22 nmol diamines (+60 nmol s.d.)/swab for the three groupsrespectively. The statistical evaluation of the three groups revealedthat the samples from the "clue cell uncertain" group were statisticallydifferent from those of the "clue cell positive" group but not fromthose of the "clue cell negative" group. Thus, the "clue cell uncertain"and "clue cell negative" groups were considered as constituting a singlegroup.

If a cut-off at 25 nmol diamines/swab is chosen as the dividing linebetween a positive and a negative result in the diamine oxidase/dyesystem assay, the sensitivity of the diamine test was 50/52=96%, i.e. 2apparent "false negatives", and the specificity was 97/119=82%, i.e. 22apparent "false positives".

"Amine" Test Comparison

Using the same cut-off value of 25 nmol diamines/swab in the diamineoxidase/dye system assay, 40 of 42 swabs judged positive in the "amine"test were also positive by the diamine test. Similarly, 13 of 15 swabsjudged possibly positive in the "amine" test and 14 of 87 swabs judgednegative for "amines" gave a positive result in the diamine test. Ifthose swabs considered possibly positive are grouped with the positives,the relative sensitivity was 53/57=93%, i.e. 4 apparent "falsenegatives", and the relative specificity 73/87=84%, i.e. 14 apparent"false positives".

The diamine oxidase/dye system results agreed well with themicrobiological culture and with the two sideroom techniques; "cluecell" and "amine" tests. It is apparent that agreements in thecomparisons with "clue cells" and the "amine" test are marginally betterthan those with the microbiological results from the Public HealthLaboratory Service.

In all three comparisons the diamine test gave slightly poorer apparentspecificity, an indication of "false positives", than sensitivity, anindication of "false negatives". Thus, sensitivity for microbiological,"clue cell" and "amine" tests was 86%, 96% and 93% respectively, whereasthe incidence of "false positives" was 21%, 18% and 16% for themicrobiological, "clue cell" and "amine" tests respectively. The closeagreement with the "amine" test might be expected because the diaminetest measures the diamines that create the characteristic odourassociated with this sideroom test.

In each of these comparisons the efficiency of the diamine assay isbeing assessed on the basis that the true answer is given by either themicrobiological culture or a sideroom test. However, no single test iscurrently recognised as a definitive test and diagnosis resides on acollective evaluation of the several tests employed in the comparisonspresented here. Thus, the evaluation of the diamine oxidase/dye systemassay indicates that it is as accurate as other recognised procedures,but has the advantages of portability and speed, and may be performed byunskilled, untrained personnel or by the patient.

EXAMPLE 8 Application of the Diamine Oxidase/Dye System to the Analysisof Diamines in the Presence of Human Serum

Procedure

Each incubation contained the following reagents:

4-aminoantipyrine (5 mg/ml; 0.1 ml);

3,5-dichloro-2-hydroxy-benzenesulphonic acid (35 mg/ml; 0.1 ml);

peroxidase (1 mg/ml; 0.1 ml);

diamine oxidase (5-15 units/ml; 0.02 ml);

putrescine or cadaverine (0, 10, 50 or 100 nmol; 0.1 ml);

human serum or buffer (0.1M tris-HCl, pH 7.5; 0.5 ml);

buffer (0.1M tris-HCl, pH 7.5) to make a final volume of 2.1 ml.

The reagents were mixed and the absorbance at 515 nm was recorded aftera 10 minute incubation. The absorbance was proportional to the amount ofputrescine or cadaverine added, so establishing that the relationshipbetween absorbance and the quantity of either putrescine or cadaverinein the assay was linear in the presence of human serum over the rangestudied (0-100 nmol diamine per assay).

                  TABLE                                                           ______________________________________                                        (undialysed serum)                                                                                  cada-                                                   putrescine                                                                            A 515 nm      verine   A 515 nm                                       nmol    -serum   +serum   nmol   -serum +serum                                ______________________________________                                        10      0.111    0.091    10     0.127  0.096                                 50      0.562    0.425    50     0.616  0.503                                 100     1.118    0.948    100    1.265  1.078                                 ______________________________________                                    

The presence of serum in the assay reduced the absorbance by 15-20%;this decrease in colour formation was the same for both putrescine andcadaverine.

Two similar series of incubations were established, one using humanserum that had been dialysed at 4° C. for 24 hours against physiologicalsaline (0.154M-NaCl), and the other using putrescine and cadaverine inthe same incubation.

                  TABLE                                                           ______________________________________                                        (dialysed serum)                                                                                    cada-                                                   putrescine                                                                            A 515 nm      verine   A 515 nm                                       nmol    -serum   +serum   nmol   -serum +serum                                ______________________________________                                        10      0.111    0.089    10     0.127  0.104                                 50      0.562    0.480    50     0.616  0.544                                 100     1.118    0.947    100    1.265  1.096                                 ______________________________________                                    

The presence of dialysed serum in the assay had a similar effect to thatexhibited by undialysed serum and the decrease in colour formation wasthe same for both putrescine and cadaverine.

In a similar manner of the findings reported in Example 2, colourformation from putrescine and cadaverine was additive in the presence ofeither dialysed or undialysed serum.

We claim:
 1. A method for the detection of putrescine and cadaverine invaginal secretions to diagnose vaginal infection comprising the stepsof:adding a sample of vaginal fluid to a mixture comprising: a diamineoxidase having a high specificity for a diamine selected from the groupconsisting of putrescine and cadaverine, and which reacts withputrescine and cadaverine to form hydrogen peroxide; and a chromogenicsystem to detect the presence of elevated levels of hydrogen peroxideresulting from the reaction between said diamine oxidase and putrescineand cadaverine.
 2. A method according to claim 1, wherein the sample isobtained by a swab.
 3. A method according to claim 1, wherein thediamine oxidase is derived from pea seedlings.
 4. A method according toclaim 1, wherein the chromogenic system is composed of peroxidase,4-aminoantipyrine, and a phenolic compound.
 5. A method according toclaim 4, wherein the phenolic compound is3,5-dichloro-2-hydroxbenzenesulphonic acid.
 6. A method according toclaim 1, wherein the vaginal infection is Gardnerella-related bacterialvaginosis.
 7. A diagnostic kit useful for the direct testing of vaginalfluid to detect the presence of elevated levels of putrescine andcadaverine for diagnosis of vaginal infection, consisting essentially ofthe following elements in at least one container:a premeasured amount ofdiamine oxidase which is highly specific for a diamine selected from thegroup consisting of putrescine and cadaverine, and which reacts withputrescine and cadaverine to form hydrogen peroxide; a premeasuredamount of a chromogenic system responsive to hydrogen peroxide; saiddiamine oxidase and said chromogenic system being disposed in said atleast one container container.
 8. The kit of claim 7 wherein saiddiamine oxidase and said chromogenic system are in freeze dried form. 9.The kit of claim 7 wherein said diamine oxidase is derived from peaseedlings.
 10. The kit of claim 7 wherein said chromogenic systemcomprises peroxidase, 4-aminoantipyrine and a phenolic compound.
 11. Thekit of claim 10 wherein the phenolic compound is3,5-dichloro-2-hydroxybenzenesulphonic acid.
 12. A diagnostic deviceuseful for the direct testing of vaginal fluid to detect the presence ofelevated levels of putrescine and cadaverine for diagnosis of vaginalinfection, consisting essentially of a paper or other surface layerabsorbed with a test composition consisting essentially of a premeasuredamount of diamine oxidase which is highly specific for a diamineselected from the group consisting of putrescine and cadaverine, andwhich reacts with putrescine and cadaverine to form hydrogen peroxide,and a premeasured amount of a chromogenic system responsive to hydrogenperoxide.
 13. The device of claim 12 wherein said diamine oxidase isderived from pea seedlings.
 14. The device of claim 12 wherein saidchromogenic system comprises peroxidase, 4-aminoantipyrine and aphenolic compound.
 15. The device of claim 14 wherein said phenoliccompound is 3,5-dichloro-2-hydroxybenzenesulphonic acid.